Tumors initiated in laboratory rodents by different chemical carcinogens can have distinctive patterns of oncogene and tumor suppressor gene mutation, suggesting that these and other genes may be critical targets of environmental carcinogens. A longstanding area of my research has been to test the hypothesis that environmental exposures produce specific patterns of gene mutation in human tumors. Such patterns can be used both to identify critical target genes and to suggest mutational mechanisms by which an environmental agent causes cancer. If specific carcinogens produce characteristic patterns of gene mutation in tumors, the detection of those patterns would be a powerful tool in studies of environmental risk and for use in prevention and early diagnosis. In a related effort, we have designed a new study to test the hypothesis that exposure correlates with the pattern of mutation in premalignant and normal lung tissues and that such mutations may have prognostic significance for lung cancer development. We are using the Lung Imaging Fluorescent Endoscope (LIFE), a newly developed bronchoscopy technique that is a very sensitive method for detecting premalignant lesions and carcinoma in situ (CIS). CIS and premalignant lesions in the lung are not resected by biopsy and there is no recognized standard-of-care treatment for such lesions. Current practice is to follow them without intervention. This will be the first systematic study to follow prospectively the natural development of lung cancer precursor lesions and CIS over time with sequential biopsies. Detection of molecular defects in such tissues may allow us to identify mutational patterns related to exposure and to provide better estimates of lung cancer risk, progression, and prognosis. Finally, we know that normal cells accumulate mutations over time and that the frequency of mutation would be expected to relect exposure, age, diet, and metabolism. Our long term goal is to develop methods that can be applied in population studies that would measure such mutations. We believe such a metric would provide a useful measure of risk for cancer, neurologic, reproductive and other diseases where DNA damage plays a role. HBV, aflatoxin and p53. Because HBV infection is often endemic in high aflatoxin exposure areas, it is still unclear whether HBV acts as a confounder or as a synergistic partner in the development of the 249ser p53 mutation. We contribute data on HCCs from southern Guangxi, a high aflatoxin exposure area. We present a metaanalysis, using our results along with those from 48 published studies, that examines the interrelationships among aflatoxin exposure, HBV infection, and p53 mutations in HCCs. We used a method that takes into account both within-study and study-to-study variability and found that the mean proportion of HCCs with the 249ser mutation was positively correlated with aflatoxin exposure (P < 0.0001). beta-Catenin. beta-Catenin plays a key role in the Wnt signaling pathway, and mutations of CTNNB1, the gene that encodes beta-catenin, have been identified in about one-fourth of human hepatocellular carcinomas from regions of low aflatoxin B1 exposure. In this study 62 hepatocellular carcinomas (HCCs) from people highly exposed to aflatoxin B1 in Guangxi, People's Republic of China, were laser-capture microdissected and examined for CTNNB1 mutations. In addition, 41 of the HCCs were evaluated for the presence of the beta-catenin protein by immunohistochemical methods. The immunohistochemical findings indicate a role for accumulation of beta-catenin and possibly increased Wnt signaling in aflatoxin B1-associated HCC. The low frequency of CTNNB1 mutations, however, suggests that mutation of another Wnt signaling component, such as the Wnt scaffolding protein axin or the adenomatous polyposis coli protein, both of which modulate beta-catenin stability, also may be involved in aflatoxin-associated HCC. DNA Polymerase beta. POL-beta is a highly conserved protein that functions in base excision repair. Loss of the POL-beta locus on chromosome 8p is a frequent event in bladder cancer, and loss of POL-beta function could hinder DNA repair leading to a mutator phenotype. Both point mutations and large intragenic deletions of POL-beta have been reported from analysis of various tumor cDNAs, but have not been investigated using genomic DNA. We noticed that the breakpoints of the presumed rearrangements were delineated by exon-exon junctions, which could instead be consistent with alternative splicing of POL-beta mRNA. We tested the hypothesis that the reported intragenic deletion were splice variants, by screening genomic DNA of human bladder tumors, bladder cancer cell lines, and normal bladder tissues for mutations or deletions in exons 1 though 14, exon a, and the promoter region of POL-beta. We found no evidence of somatic mutations or deletions in our sample set, although two polymorphisms were identified. Examination of cDNA from a subset of the original sample set revealed that truncated forms of POL-beta were surprisingly common. 48 of 89 (54%) of sequenced cDNA clones had large deletions, each beginning and/or ending exactly at exon-exon junctions. Since these deletions occur at exon-exon junctions and are seen in cDNA but not genomic DNA, they are consistent with alternative mRNA splicing. We described 12 different splicing events, occurring in 18 different combinations. Loss of exon 2 was the most frequent, being found in 42 of 49 (86%) of the variant sequenced clones. Mutational Hotspots in the Human Genome: Sporadic microsatellite mutations are frequently observed in lung, bladder and head and neck tumors with intact DNA mismatch repair. AAAG tetranucleotide repeats appear to be especially prone to the accumulation of these mutations. We hypothesized that occurrences of microsatellite mutations in these cancers may be linked to DNA damage caused by exposure to carcinogens in tobacco smoke. To test this hypothesis, we developed a model system based on reactivation of Green Fluorescent Protein (GFP) in which a plasmid vector carries a microsatellite repeat that places the GFP sequence out of frame for protein translation. In this reporter system, DNA slippage mutations can restore the GFP reading frame and become detectable by flow cytometry as GFP-positive cells. Pools of stably transfected RKO cells were treated at four dose levels each of g-irradiation, BPDE, MNNG, t-BHP, and UV-irradiation and assayed for GFP-positive cells 48 hours later. We studied the microsatellite repeats AAAG, ATAG, CAGT, and CA, as well as a control sequence lacking any repetitive elements. A log-linear regression approach was used to discriminate between the effects of repeat unit and dose for each agent. A statistically significant increase in GFP-positive cells was found with increasing dose with all agents, although repeat-unit specific response patterns were only observed with MNNG, t-BHP and UV-irradiation. With MNNG, significant differences in response were observed between dinucleotide and tetranucleotide repeat units. The effects of UV-irradiation were consistent with the predicted number of pyrimidine dimers per repeat unit, with higher GFP activation in repeats that had large numbers of adjacent pyrimidines. We found no evidence to indicate that AAAG repeat responded to any of the DNA damaging agents with higher levels of GFP activation than other repeat units.